Concurrent positional dynamics and activity mapping of DNA-binding proteins.

Single-molecule techniques have advanced our understanding of DNA-protein interactions, yet simultaneously tracking enzyme position on DNA, determining binding dynamics and resolving enzyme activity remained challenging. Here, we present a protocol for concurrent positional dynamics and activity mapping (C-DAM) of DNA-binding proteins that integrates optical tweezers and confocal fluorescence microscopy to achieve concurrent, high-resolution correlation of enzyme activity with its precise location on DNA. The protocol uses optical tweezers to track enzymes converting DNA between single- and double-stranded forms, measuring mechanical changes to resolve enzymatic activity at nanometer spatial and millisecond temporal resolution. This mechanical information is then correlated with simultaneously acquired, diffraction-limited fluorescence signals through 'force-assisted imaging', which pinpoints fluorescently labeled molecules relative to their enzymatic activity site. Unlike existing methods analyzing mechanical and fluorescence data separately, C-DAM leverages both techniques synergistically, providing direct, concurrent mapping of enzyme position, binding dynamics and catalytic activity. The protocol enables dissection of distinct functional states, rapid enzyme exchange events and location-specific behaviors, particularly for multifunctional proteins at dynamic single-stranded DNA (ssDNA)-double-stranded DNA (dsDNA) junctions. By correlating DNA and protein kymographs, the method distinguishes different binding modes, such as static binding to ssDNA versus diffusion on dsDNA, providing quantitative information on protein-DNA interactions. The protocol is broadly adaptable for investigating DNA-binding proteins and their complexes, with potential for future integration with techniques like Förster resonance energy transfer. Excluding sample preparation and instrument construction, data acquisition and analysis for a typical dataset takes 2-6 h and requires expertise in single-molecule biophysics, programming and data analysis.